6.1.1. Biology and life cycle of
Daphnia

Daphnia is a frequently used food source in the
freshwater larviculture (i.e. fordifferent carp species) and in the
ornamental fish industry (i.e.guppies, sword tails, black mollies and
plattys etc.)

Daphnia belongs to the suborder Cladocera, which are
small crustaceans that are almost exclusively living in freshwater. The carapace
encloses the whole trunk, except the head and the apical spine (when present).
The head projects ventrally and somewhat posteriorly in a beak-like snout. The
trunk appendages (five or six pairs) are flattened, leaf-like structures that
serve for suspension feeding (filter feeders) and for locomotion. The anterior
part of the trunk, the postabdomen is turned ventrally and forward and bears
special claws and spines to clean the carapace (Fig. 6.1.). Species of the genus
Daphnia are found from the tropics to the arctic, in habitats varying in
size from small ponds to large freshwater lakes. At present 50 species of
Daphnia are reported worldwide, of which only six of them normally occur
in tropical lowlands.

The adult size is subjected to large variations; when food is
abundant, growth continues throughout life and large adults may have a carapace
length twice that of newly-mature individuals. Apart from differences in size,
the relative size of the head may change progressively from a round to
helmet-like shape between spring and midsummer. From midsummer to fall the head
changes back to the normal round shape. These different forms are called
cyclomorphs and may be induced, like in rotifers, by internal factors, or may be
the result from an interaction between genetic and environmental
conditions.

Normally there are 4 to 6 Instar stages; Daphnia
growing from nauplius to maturation through a series of 4-5 molts, with the
period depending primarily on temperature (11 days at 10°C to 2 days at
25°C) and the availability of food. Daphnia species reproduce either
by cyclical or obligate parthenogenesis and populations are almost exclusively
female. Eggs are produced in clutches of two to several hundred, and one female
may produce several clutches, linked with the molting process. Parthenogenetic
eggs are produced ameiotically and result in females, but in some cases males
can appear. In this way the reproductive pattern is similar to rotifers, where
normally parthenogenetic diploid eggs are produced. The parthenogenetic eggs
(their number can vary from 1 to 300 and depends largely upon the size of the
female and the food intake) are laid in the brood chamber shortly after ecdysis
and hatch just before the next ecdysis. Embryonic development in cladocerans
occurs in the broodpouch and the larvae are miniature versions of the adults. In
some cases the embryonic period does not correspond with the brood period, and
this means that the larvae are held in the brood chamber even after the
embryonic period is completed, due to postponed ecdysis (environmental factors).
For different species the maturation period is remarkably uniform at given
temperatures, ranging from 11 days at 10°C to only 2 days at
25°C.

Factors, such as change in water temperature or food
depreviation as a result of population increase, may induce the production of
males. These males have one or two gonopores, which open near the anus and may
be modified into a copulatory organ. The male clasps the female with the first
antennae and inserts the copulatory processes into the single, median female
gonopore. The fertilized eggs are large, and only two are produced in a single
clutch (one from each ovary), and are thick-shelled: these resting or dormant
eggs being enclosed by several protective membranes, the ephippium. In this
form, they are resistant to dessication, freezing and digestive enzymes, and as
such play an important role in colonizing new habitats or in the
re-establishment of an extinguished population after unfavourable seasonal
conditions.

6.1.2. Nutritional value of
Daphnia

The nutritional value of Daphnia depends strongly on
the chemical composition of their food source. However, since Daphnia is
a freshwater species, it is not a suitable prey organism for marine organisms,
because of its low content of essential fatty acids, and in particular(n-3) HUFA. Furthermore, Daphnia contains a broad spectrum of
digestive enzymes such, as proteinases, peptidases, amylases, lipases and even
cellulase, that can serve as exo-enzymes in the gut of the fish
larvae.

6.1.3. Feeding and nutrition of
Daphnia

The filtering apparatus of Daphnia is constructed of
specialized thoracic appendages for the collection of food particles. Five
thoracic limbs are acting as a suction and pressure pump. The third and fourth
pair of appendages carry large filter-like screens which filter the particles
from the water. The efficiency of the filter allows even the uptake of bacteria
(approx. 1µm). In a study on the food quality of freshwater phytoplankton
for the production of cladocerans, it was found that from the spectrum
blue-greens, flagellates and green algae, Daphnia performed best on a
diet of the cryptomonads, Rhodomonas minuta and Cryptomonas sp.,
containing high levels of HUFA (more than 50% of the fatty acids in these two
algae consisted of EPA and DHA, while the green algae were characterized by more
18:3n-3). This implies that the long-chained polyunsaturated fatty acids are
important for a normal growth and reproduction of Daphnia. Heterotrophic
microflagellates and ciliates up to the size of Paramecium can also be
used as food for Daphnia. Even detritus and benthic food can be an
important food source, especially when the food concentration falls below a
certain threshold. In this case, the water current produced by the animals
swimming on the bottom whirls up the material which is eventually ingested.
Since daphnids seem to be non-selective filter feeders (i.e., they do not
discriminate between individual food particles by taste) high concentrations of
suspended material can interfere with the uptake of food particles.

Figure 6.1. Schematic drawing of
the internal and external anatomy of Daphnia.

6.1.4. Mass culture of
Daphnia

6.1.4.1. General procedure for tank
culture

Daphnia is very sensitive to contaminants, including
leaching components from holding facilities. When plastic or other polymer
containers are used, a certain leaching period will be necessary to eliminate
toxic compounds.

The optimal ionic composition of the culture medium for
Daphnia is unknown, but the use of hard water, containing about 250
mg.l-1 of CO32-, is recommended. Potassium and
magnesium levels should be kept under 390 mg.l-1 and 30-240 µg.
l-1, respectively. Maintenance of pH between 7 to 8 appears to be
important to successful Daphnia culture. To maintain the water hardness
and high pH levels, lime is normally added to the tanks. The optimal culture
temperature is about 25°C and the tank should be gently aerated to keep
oxygen levels above 3.5 mg.l-1 (dissolved oxygen levels below 1.0
mg.l-1 are lethal to Daphnia). Ammonia levels must be kept
below 0.2 mg.l-1.

Inoculation is carried out using adult Daphnia or
resting eggs. The initial density is generally in the order of 20 to 100 animals
per litre.

Normally, optimal algal densities for Daphnia culture
are about 105 to 106 cells. ml-1 (larger
species of Daphnia can support 107 to 109
cells.ml-1). There are two techniques to obtain the required algal
densities: the detrital system and the autotrophic system:

6.1.4.2. Detrital system

The stable tea rearing system is a culture medium
made up of a mixture of soil, manure and water. The manure acts as a fertilizer
to promote algal blooms on which the daphnids feed. One can make use of fresh
horse manure (200 g) that is mixed with sandy loam or garden soil (1 kg) in 10 l
pond water to a stable stock solution; this solution diluted two to four times
can then be used as culture medium. Other fertilizers commonly used are: poultry
manure (4 g.l-1) or cow-dung substrates. This system has the
advantage to be self-maintaining and the Daphnia are not quickly
subjected to deficiencies, due to the broad spectrum of blooming algae. However,
the culture parameters in a detrital system are not reliable enough to culture
Daphnia under standard conditions, i.e. overfertilization may occur,
resulting in anoxic conditions and consequently in high mortalities and/or
ephippial production.

6.1.4.3. Autotrophic
system

Autotrophic systems on the other hand use the addition of
cultured algae. Green water cultures (105 to 106
cells.ml-1) obtained from fish pond effluents are frequently used but
these systems show much variation in production rate mainly because of the
variable composition of algal species from one effluent to another. Best control
over the culture medium is obtained when using pure algal cultures. These can be
monocultures of e.g. algae such as Chlorella, Chlamydomonas
or Scenedesmus, or mixtures of two algal cultures. The problem with these
selected media is that they are not able to sustain many Daphnia
generations without the addition of extra vitamins to the Daphnia
cultures. A typical vitamin mix is represented in Table 6.1.

Table 6.1. A vitamin mix for the monospecific culture of
Daphnia on Selenastrum, Ankistrodesmus or Chlamydomonas. One
ml of this stock solution has to be added to each litre of algal culture medium
(Goulden et al., 1982).

Nutrient

Concentration of stock solution
(µg.1-1)

Biotin

5

Thiamine

100

Pyridoxine

100

Pyridoxine

3

Calcium Panthothenate

250

B12 (as mannitol)

100

Nicotinic acid

50

Nicotinomide

50

Folic acid

20

Riboflavin

30

Inositol

90

To calculate the daily algal requirements and to estimate the
harvesting time, regular sampling of the population density must be routinely
undertaken. Harvesting techniques can be non-selective irrespective of size or
age group, or selective (only the medium sized daphnids are harvested, leaving
the neonates and matured individuals in the culture tank).

Mass cultivation of Daphnia magna can also be achieved
on cheap agro-industrial residues, like cotton seed meal (17 g.l-1),
wheat bran (6.7 g.l-1), etc. Rice bran has many advantages in
comparison to other live foods (such as microalgae): it is always available in
large quantities, it can be purchased easily at low prices, it can be used
directly after simple treatment (micronisation, defatting), it can be stored for
long periods, it is easy to dose, and it has none of the problems involved in
maintenance of algal stocks and cultures.

In addition to these advantages, there is also the fact that
rice bran has a high nutritional value; rice bran (defatted) containing 24%
(18.3%) crude protein, 22.8% (1.8%) crude fat, 9.2% (10.8%) crude fibre, and
being a rich source of vitamins and minerals. Daphnia can be grown on
this food item for an unlimited number of generations without noticeable
deficiencies.

Defatted rice bran is preferred above raw rice bran because it
prevents hydrolysis of the fatty acids present and, consequently, rancidity of
the product. Micronisation of the bran into particles of less than 60 µm is
generally carried out by treating an aqueous suspension (50 g.l-1)
with a handmixer and filtering it through a 60 µm sieve, or by preparing it
industrially by a dry mill process. The suspension is administered in small
amounts throughout a 24 h period: 1 g of defatted rice bran per 500 individuals
for two days (density: 100 animals.l-1). The food conversion ratio
has an average of 1.7, which implies that with less than 2 kg of dry rice bran
approximately 1 kg wet daphnid material can be produced (with a 25% water
renewal per week; De Pauw et al., 1981).

6.1.4.4. General procedure for pond
culture

Daphnia can also be produced in ponds of at least 60 cm
in height. To produce 1 ton of Daphnia biomass per week, a 2500
m3 culture pond is required. The pond is filled with 5 cm of
sun-dried (for 3 days) soil to which lime powder is added at a rate of 0.2 kg
lime powder per ton soil. After this the pond is then filled with water up to 15
cm. Poultry manure is added to the ponds on the 4th day at a rate of 0.4
kg.m-3 to promote phytoplankton blooms. Fertilization of the pond
with organic manure instead of mineral fertilizers is preferred because
cladocerans can utilize much of the manure directly in the form of detritus. On
day 12 the water level is raised to 50 cm and the pond is fertilized a second
time with poultry manure (1 kg.m-3). Thereafter, weekly fertilization
rates are maintained at 4 kg poultry manure per m-3. In addition,
fresh cow dung may also be used: in this instance a suspension is prepared
containing 10 g.l-1, which is then filtered through a 100 µm
sieve. During the first week a 10 l extract is used per day per ton of water;
the fertilization increasing during the subsequent weeks from 20
l.m-3.day-1 in the second week to 30
l.m-3.day-1 in the following weeks.

The inoculation of the ponds is carried out on the 15th day at
a rate of 10 daphnids per litre. One month after the inoculation, blooms of more
than 100 g.m-3 can be expected. To maintain water quality in these
ponds, fresh hard water can be added at a maximum rate of 25% per day.
Harvesting is carried out by concentrating the daphnids onto a 500 µm
sieve. The harvested biomass is concentrated in an aerated container (< 200
daphnids.l-1). In order to separate the daphnids from unfed
substrates, exuviae and faecal material, the content of the container is brought
onto a sieve, which is provided with a continuous circular water flow. The unfed
particles, exuviae and faeces will collect in the centre on the bottom of the
sieve, while the daphnids remain in the water column. The unwanted material can
then be removed by using a pipette or sucking pump. Harvesting can be complete
or partial; for partial harvesting a maximum of 30% of the standing crop may be
harvested daily.

6.1.4.5. Contamination

Daphnia cultures are often accidentally contaminated
with rotifers. In particular Brachionus, Conochilus and some bdelloids
may be harmful, (i.e. B. rubens lives on daphnids and hinders swimming
and food collection activities). Brachionus is simply removed from the
culture by flushing the water and using a sieve of appropriate mesh size as
Daphnia is much bigger than Brachionus. Conochilus, on the
other hand, can be eliminated by adding cow dung to the culture (lowering the
oxygen levels). Bdelloids are more difficult to remove from the culture since
they are resistant to a wide range of environmental conditions and even drought.
However, elimination is possible by creating strong water movements, which bring
the bdelloids (which are bottom dwellers) in the water column, and then removing
them by using sieves.

6.1.5. Production and use of resting
eggs

Resting eggs are interesting material for storage, shipment
and starting of new Daphnia cultures. The production of resting eggs can
be initiated by exposing a part of the Daphnia culture to a combination
of stressful conditions, such as low food availability, crowding of the animals,
lower temperatures and short photoperiods. These conditions are generally
obtained with aging populations at the end of the season. Collection of the
ephippia from the wild can be carried out by taking sediment samples, rinsing
them through a 200 µm sieve and isolating the ephippia under a binocular
microscope. Normally, these embryos remain in dormancy and require a diapause
inhibition to terminate this status, so that they can hatch when conditions are
optimal. Possible diapause termination techniques are exposing the ephippia to
low temperatures, darkness, oxygen and high carbon dioxide concentrations for a
minimal period of several weeks (Davison, 1969).

There is still no standard hatching procedure for Daphnia.
Generally the hatching process is stimulated by exposing the ephippia to higher
temperatures (17-24°C), bright white light (70 W.m-2), longer
photoperiods and high levels of dissolved oxygen. It is important, however, that
these shocks are given while the resting eggs are still in the ephippium. After
the shock the eggs may be removed from the ephippium. The hatching will then
take place after 1-14 days.

6.1.6. Use of Moina

Moina also belongs to the Cladocera and many of the
biological and cultural characteristics that have been discussed for
Daphnia can be applied to Moina.

Moina thrives in ponds and reservoirs but primarily
inhabits temporary ponds or ditches. The period to reach reproductive maturity
takes four to five days at 26°C. At maturity clear sexual dimorphic
characteristics can be observed in the size of the animals and the antennule
morphology. Males (0.6-0.9 mm) are smaller than females (1.0-1.5 mm) and have
long graspers which are used for holding the female during copulation. Sexually
mature females carry only two eggs enclosed in an ephippium which is part of the
dorsal exoskeleton.

Moina is of a smaller size than Daphnia, with a
higher protein content, and of comparable economic value. Produced biomass is
successfully used in the larviculture of rainbow trout, salmon, striped bass and
by tropical fish hobbyists who also use it in a frozen form to feed over sixty
fresh and salt water fish varieties. The partial replacement of Artemia
by Moina micrura was also reported to have a positive effect during the
larviculture of the freshwater prawn Macrobrachium rosenbergii (Alam,
1992).

Enrichment of Moina can be carried out using the direct
method, by culturing them on bakers yeast and emulsified fish or
cuttlefish liver oils. Experiments have shown that Moina takes up (n-3)
HUFA in the same way, although slower, than rotifers and Artemia nauplii,
reaching a maximum concentration of around 40% after a 24 h-feeding
period.